Inhibition of Mitochondria- and Endoplasmic Reticulum Stress-Mediated Autophagy Augments Temozolomide-Induced Apoptosis in Glioma Cells

Autophagy is a crucial process for cells to maintain homeostasis and survival through degradation of cellular proteins and organelles, including mitochondria and endoplasmic reticula (ER). We previously demonstrated that temozolomide (TMZ), an alkylating agent for brain tumor chemotherapy, induced reactive oxygen species (ROS)/extracellular signal-regulated kinase (ERK)-mediated autophagy to protect glioma cells from apoptosis. In this study, we investigated the role of mitochondrial damage and ER stress in TMZ-induced cytotoxicity. Mitochondrial depolarization and mitochondrial permeability transition pore (MPTP) opening were observed as a prelude to TMZ-induced autophagy, and these were followed by the loss of mitochondrial mass. Electron transport chain (ETC) inhibitors, such as rotenone (a complex I inhibitor), sodium azide (a complex IV inhibitor), and oligomycin (a complex V inhibitor), or the MPTP inhibitor, cyclosporine A, decreased mitochondrial damage-mediated autophagy, and therefore increased TMZ-induced apoptosis. TMZ treatment triggered ER stress with increased expression of GADD153 and GRP78 proteins, and deceased pro-caspase 12 protein. ER stress consequently induced autophagy through c-Jun N-terminal kinases (JNK) and Ca2+ signaling pathways. Combination of TMZ with 4-phenylbutyrate (4-PBA), an ER stress inhibitor, augmented TMZ-induced cytotoxicity by inhibiting autophagy. Taken together, our data indicate that TMZ induced autophagy through mitochondrial damage- and ER stress-dependent mechanisms to protect glioma cells. This study provides evidence that agents targeting mitochondria or ER may be potential anticancer strategies

Evodiamine Induces Transient Receptor Potential Vanilloid-1-Mediated Protective Autophagy in U87-MG Astrocytes

Cerebral ischemia is a leading cause of mortality and morbidity worldwide, which results in cognitive and motor dysfunction, neurodegenerative diseases, and death. Evodiamine (Evo) is extracted from Evodia rutaecarpa Bentham, a plant widely used in Chinese herbal medicine, which possesses variable biological abilities, such as anticancer, anti-inflammation, antiobesity, anti-Alzheimer’s disease, antimetastatic, antianoxic, and antinociceptive functions. But the effect of Evo on ischemic stroke is unclear. Increasing data suggest that activation of autophagy, an adaptive response to environmental stresses, could protect neurons from ischemia-induced cell death. In this study, we found that Evo induced autophagy in U87-MG astrocytes. A scavenger of extracellular calcium and an antagonist of transient receptor potential vanilloid-1 (TRPV-1) decreased the percentage of autophagy accompanied by an increase in apoptosis, suggesting that Evo may induce calcium-mediated protective autophagy resulting from an influx of extracellular calcium. The same phenomena were also confirmed by a small interfering RNA technique to knock down the expression of TRPV1. Finally, Evo-induced c-Jun N-terminal kinases (JNK) activation was reduced by a TRPV1 antagonist, indicating that Evo-induced autophagy may occur through a calcium/c-Jun N-terminal kinase (JNK) pathway. Collectively, Evo induced an influx of extracellular calcium, which led to JNK-mediated protective autophagy, and this provides a new option for ischemic stroke treatment

Evodiamine Induces Transient Receptor Potential Vanilloid-1-Mediated Protective Autophagy in U87-MG Astrocytes

Cerebral ischemia is a leading cause of mortality and morbidity worldwide, which results in cognitive and motor dysfunction, neurodegenerative diseases, and death. Evodiamine (Evo) is extracted from Evodia rutaecarpa Bentham, a plant widely used in Chinese herbal medicine, which possesses variable biological abilities, such as anticancer, anti-inflammation, antiobesity, antiAlzheimer's disease, antimetastatic, antianoxic, and antinociceptive functions. But the effect of Evo on ischemic stroke is unclear. Increasing data suggest that activation of autophagy, an adaptive response to environmental stresses, could protect neurons from ischemia-induced cell death. In this study, we found that Evo induced autophagy in U87-MG astrocytes. A scavenger of extracellular calcium and an antagonist of transient receptor potential vanilloid-1 (TRPV-1) decreased the percentage of autophagy accompanied by an increase in apoptosis, suggesting that Evo may induce calcium-mediated protective autophagy resulting from an influx of extracellular calcium. The same phenomena were also confirmed by a small interfering RNA technique to knock down the expression of TRPV1. Finally, Evo-induced c-Jun N-terminal kinases (JNK) activation was reduced by a TRPV1 antagonist, indicating that Evo-induced autophagy may occur through a calcium/c-Jun N-terminal kinase (JNK) pathway. Collectively, Evo induced an influx of extracellular calcium, which led to JNK-mediated protective autophagy, and this provides a new option for ischemic stroke treatment

ETC inhibitors suppress TMZ-induced ROS generation.

<p>U87 MG cells were pretreated with or without 20 nM rotenone, 150 µM sodium azide, or 1 nM oligomycin for 1 h followed by incubation with 400 µM TMZ for 36 h, and ROS levels were analyzed by HEt, DCFH-DA, and DHR123 staining using flow cytometry. Rot, rotenone; NaN₃, sodium azide; Oligo, oligomycin. Results are presented as the mean ± SD. *<i>p</i><0.05, **<i>p</i><0.01 vs. each respective TMZ group.</p

ETC inhibitors reduce TMZ-induced autophagy and increase apoptosis and cell death.

<p>U87 MG cells were treated with or without 20 nM rotenone, 150 µM sodium azide, or 1 nM oligomycin for 1 h followed by incubation with 400 µM TMZ for another 72 h. Autophagy (A) and apoptosis (B) were assessed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038706#s4" target="_blank">Materials and Methods</a>”. (C) U87 MG cells were pretreated with ETC inhibitors such as rotenone, sodium azide, and oligomycin for 1 h, followed by treatment with TMZ for 36 h. Cell lysates were analyzed by immunoblotting using antibodies against PARP, caspase 3, LC3, and GAPDH. GAPDH was used as an internal control to normalize the amount of proteins applied in each lane. Cells were treated as in (A) and were analyzed by MTT assay (D), LDH release assay (E), and colony formation assay (F). Results are presented as the mean±SD. Rot, rotenone; NaN₃, sodium azide; Oligo, oligomycin. **<i>p</i><0.01 vs. each respective TMZ group.</p

TMZ induces mitochondrial depolarization, MPTP opening, and loss of mitochondrial mass.

<p>U87 MG cells were treated with 400 µM TMZ for the indicated time course. The mitochondrial membrane potential (A and B), MPTP (C and D), and mitochondrial mass (E and F) were measured using flow cytometry with rhodamine 123, calcein AM/CoCl₂, and NAO staining, respectively. Data presented in panels (A), (C), and (E) are representative of three independent experiments, and their statistical results are presented as the mean ± SD in panels (B), (D), and (F), respectively. *<i>p</i><0.05, **<i>p</i><0.01 vs. each respective control.</p

TMZ triggers ER stress to induce autophagy.

<p>(A and B) U87 MG cells were treated with 400 µM TMZ for the indicated time periods and then analyzed by immunoblotting with anti-GADD153, anti-GRP78, anti-GAPDH (A), and anti-caspase 12 (B) antibodies. (C) U87 MG cells were pretreated with 10 mM 4-PBA for 1 h, followed by treatment with TMZ for 36 h (for analysis of GRP78, GADD153, and LC3) or 72 h (for analysis of PARP, caspase 12, and caspase 3). Cell lysates were analyzed by immunoblotting. GAPDH was used as an internal control to normalize the amount of proteins applied in each lane. (D) U87 MG cells were treated as in (C) for 72 h to determine percentages of cells undergoing autophagy and apoptosis, as well as the cell viability (E) and percent of LDH release (F). (G) Long-term viability of cells after TMZ and 4-PBA co-treatment was measured by colony formation assay. Results are presented as the mean ± SD. **<i>p</i><0.01 vs. each respective control. ^##<i>p</i><0.01 vs. each respective TMZ group.</p

Effects of the autophagy inhibitor, 3-MA on the mitochondrial membrane potential and mitochondrial mass.

<p>U87 MG cells were pre-treated with or without 2 mM 3-MA for 1 h followed by incubation with 400 µM TMZ for 36 h or 72 h. The mitochondrial membrane potential (A) and mitochondrial mass (B) were analyzed using flow cytometry with rhodamine 123 and NAO staining, respectively. Results are presented as the mean ± SD. **<i>p</i><0.01 vs. each respective TMZ group.</p

The ETC inhibitors reverse TMZ-induced mitochondrial depolarization and the loss of mitochondrial mass.

<p>(A and B) U87 MG cells were pre-treated with or without 20 nM rotenone, 150 µM sodium azide, or 1 nM oligomycin for 1 h followed by incubation with 400 µM TMZ for 36 h, and then the mitochondrial membrane potential was analyzed using flow cytometry with rhodamine 123. (C) Mitochondrial mass was detected with NAO staining after the same treatment for 72 h. Data presented in panel (A) are representative of three independent experiments, and their statistical results are presented as the mean ± SD in panel (B). Rot, rotenone; NaN₃, sodium azide; Oligo, oligomycin. **<i>p</i><0.01 vs. each respective TMZ group.</p

ER stress induces intracellular Ca²⁺ levels after TMZ treatment.

<p>(A) U87 MG cells were treated with 400 µM TMZ or combined with 10 mM 4-PBA or 5 µM BAPTA-AM/2 mM EGTA for the indicated time periods. Then the level of intracellular Ca²⁺ was measured using flow cytometry with Fluo-3 AM staining. U87 MG cells were pretreated with the indicated concentrations of 5 µM BAPTA-AM/2 mM EGTA for 1 h, followed by treatment with 400 µM TMZ for another 72 h to determine percentages of cells undergoing autophagy and apoptosis (B), as well as the cell viability (C) and percent of LDH release (D). (E) Colony formation was measured after TMZ and BAPTA-AM/EGTA co-treatment. Results are presented as the mean ± SD. **<i>p</i><0.01 vs. each respective control. ^##<i>p</i><0.01 vs. each respective TMZ group.</p